GreA

ABSTRACT

The invention provides greA polypeptides and DNA (RNA) encoding greA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing greA polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of Provisional Patent Application, Ser.No. 60/053,639, filed Jul. 24, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the gre (transcript cleavage factor) family, hereinafterreferred to as "greA".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome The frequency of Staphylococcus aureus infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This has created a demand forboth new anti-microbial agents and diagnostic tests for this organism.

Transcription of DNA is often arrested at sites in DNA that trap afraction of elongating RNA polymerase molecules that pass through,resulting in blocked ternary complexes that cannot propagate ordissociate their RNA product. Transcript cleavage factors induce RNApolymerase to cleave the RNA in such complexes at the 3'-end, allowingRNA polymerase to back up and re-attempt to read through the potentialtrap (Borukhov et al. 1993. Cell 72:459-466). In addition to assuringefficient transcript elongation, transcript cleavage factors increasethe fidelity of transcription since misincorporated bases at the 3'-endof the nascent RNA also lead to arrested complexes (Erie et al. Science262:867-8730. Two transcript cleavage factors, GreA and GreB, have beenidentified in E. coli (Borukhov et al. 1993. Cell 72:459-466).GreA-dependent transcript cleavage usually results in the removal of di-and trinucleotides from the 3'-end of the stalled RNA. GreB-dependentcleavage yields larger oligonucleotides, up to a length of ninenucleotides. Both proteins bind RNA polymerase. Neither the GreA or GreBproteins possess intrinsic nuclease activity rather they stimulate anuclease activity inherent in RNA polymerase (Orlova et al. 1995. Proc.Natl. Acad. Sci., USA 92:4596-4600). The GreA and GreB proteins arehomologous, sharing 38% sequence identity (59% similar). The eukaryotictranscript elongation protein SII is similar to the GreA and GreBproteins in that it stimulates RNA cleavage from the 3'-end of RNA in astalled complex but does not share significantly sequence homology withthe GreA and GreB proteins (Borukhov et al. 1993. Cell 72:459-466).

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfiinctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown E. coli greA protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel greA polypeptides by homology between the amino acidsequence set out in Table 1 SEQ ID NO:2! and a known amino acid sequenceor sequences of other proteins such as E. coli greA protein.

It is a further object of the invention to provide polynucleotides thatencode greA polypeptides, particularly polynucleotides that encode thepolypeptide herein designated greA.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding greA polypeptides comprisingthe sequence set out in Table 1 SEQ ID NO:1! which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel greA protein from Staphylococcus aureus comprising the amino acidsequence of Table 1 SEQ ID NO:2!, or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding greA, particularly Staphylococcus aureus greA,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of greA and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as greA as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of greA polypeptide encoded by naturally occurring alleles ofthe greA gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned greA polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing greAexpression, treating disease, for example, disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa greA polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to greA polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against greA polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided greA agonists and antagonists, preferably bacteriostatic orbactericidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a greA polynucleotide or a greA polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO:1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO:1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO:2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzyrmatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.

"Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s). "Polypeptide(s)" refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. "Polypeptide(s)" refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers and to longer chains generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene encodedamino acids. "Polypeptide(s)" include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. It will be appreciated that thesame type of modification may be present in the same or varying degreeat several sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. Modifications include,for example, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS--STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel greA polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel greA of Staphylococcusaureus, which is related by amino acid sequence homology to E. coli greApolypeptide. The invention relates especially to greA having thenucleotide and amino acid sequences set out in Table 1 SEQ ID NO:1! andTable 1 SEQ ID NO:2! respectively, and to the greA nucleotide sequencesof the DNA in the deposited strain and amino acid sequences encodedthereby.

                                      TABLE 1    __________________________________________________________________________    greA Polynucleotide and Polypeptide Sequences    __________________________________________________________________________    (A) Sequences from Staphylococcus aureus greA polynucleotide sequence     SEQ ID NO:1!.     ##STR1##    (B) greA polypeptide sequence deduced from the polynucleotide sequence in    this table  SEQ ID NO:2!.     ##STR2##    (C) Polynucleotide sequence embodiments  SEQ ID NO:1!.     ##STR3##    (D) Polypeptide sequence embodiments  SEQ ID NO:2!.     ##STR4##    (E) Sequences from Staphylococcus aureus greA polynucleotide ORF sequence     SEQ ID NO:3!.     ##STR5##    (F) greA polypeptide sequence deduced from the polynucleotide ORF    sequence in this table  SEQ ID NO:4!.     ##STR6##    __________________________________________________________________________

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Sep., 1995 and assigned NCIMB Deposit No. 40771, and isreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length greA gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 SEQID NO:2! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofgreA, and also those which have at least 70% identity to a polypeptideof Table 1 SEQ ID NOS:2 and 4! or the relevant portion, preferably atleast 80% identity to a polypeptide of Table 1 SEQ ID NOS:2 and 4!, andmore preferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) SEQ ID NO:2! wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with greA polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 SEQ ID NOS:2 and 4!, orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of greA, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the greA polypeptide havinga deduced amino acid sequence of Table 1 SEQ ID NOS:2 and 4! andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 SEQ ID NOS:1 and 3!, a polynucleotide of theinvention encoding greA polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 SEQ ID NOS: 1 and 3!,typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 SEQ ID NO:1! was discovered in a DNA library derived fromStaphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 SEQ ID NOS: 1! contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 SEQ ID NO:2! with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO:1, betweennucleotide number 1 through number 474 encodes the polypeptide of SEQ IDNO:2. The stop codon begins at nucleotide number 475 of SEQ ID NO:1.

The greA protein of the invention is structurally related to otherproteins of the gre (transcript cleavage factor) family, as shown by theresults of sequencing the DNA encoding greA of the deposited strain. Theprotein exhibits greatest homology to E. coli greA protein among knownproteins. The greA protein of Table 1 SEQ ID NO:2! has about 46%identity over its entire length and about 64% similarity over its entirelength with the amino acid sequence of E. coli greA polypeptide. Theaccession numbers of related sequences are found as follows: U01376 (E.coli greA); U32812 (H. influenzae greA, presumptive); Z12122 (Rickettsiaprowazekii greA, presumptive); U39708 (Mycoplasma genitalium greA,presumptive); AE000043 (Mycoplasma pneumoniae greA, presumptive); U81522(E. coli greB); U32738 (H. influenzae greB, presumptive) U15183(Mycobacterium leprae greA, presumptive);

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 SEQ ID NO:1!. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et aL,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to 474 or 477 set forth in SEQ ID NO:1 of Table1 which encode the greA polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) SEQ ID NO: 1! wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₂ and R₁ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus greA havingthe amino acid sequence set out in Table 1 SEQ ID NO:2!. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 SEQ ID NO:2!. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodinggreA variants, that have the amino acid sequence of greA polypeptide ofTable 1 SEQ ID NO:2! in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof greA.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding greA polypeptide having an amino acid sequence set out in Table1 SEQ ID NOS:2 and 4!, and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding greA polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding greA and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the greA gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the greA gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO:1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotides of the invention may encode a mature protein,a mature protein plus a leader sequence (which may be referred to as aproprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the greA polynucleotides ofthe invention for use as diagnostic reagents. Detection of greA in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the greA genc may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled greA polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding greA can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

                  TABLE 2    ______________________________________    Primers for amplification of greA polynucleotides    SEQ    ID NO  PRIMER SEQUENCE    ______________________________________    5      5'-GGAATTCATGGAAAATCACAAGCAATATCCAAT-3'    6      5'-GGAATTCATCATACTCAGAGTTCTCTGATAAGTC-3'    ______________________________________

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying greA DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 SEQ ID NO:1!. Increased or decreasedexpression of greA polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of greA protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a greAprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-greA or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against greA- polypeptide may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J. Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of greApolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising greA polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a greA agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the greA polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of greA polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in greA polynucleotides or polypeptide activity,and binding assays known in the art.

Another example of an assay for greA antagonists is a competitive assaythat combines greA and a potential antagonist with greA-bindingmolecules, recombinant greA binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. The greA protein can be labeled,such as by radioactivity or a colorimetric compound, such that thenumber of greA molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing greA-induced activities, thereby preventing the action of greAby excluding greA from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of greA.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock greA protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial greA proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofgreA) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with greA, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of greA, or afragment or a variant thereof, for expressing greA, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a greA or protein coded therefrom,wherein the composition comprises a recombinant greA or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid greA or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+T cells.

A greA polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain greAprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg.

The physician in any event will determine the actual dosage which willbe most suitable for an individual and will vary with the age, weightand response of the particular individual. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 6    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 477 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGGAAAATCACAAGCAATATCCAATGACTCAAGAAAGTTTTGAAAAATTAGAGCGTGAA60    CTTGAAGAATTAAAAACAGTTAAGCGTCCTGAAGTTGTAGAGAAAATTAAAGTTGCACGT120    TCATTTGGTGACTTATCAGAGAACTCTGAGTATGATGCAGCAAAAGATGAACAAGGATTC180    ATCGAACAAGATATTCAAAGAATTGAGCATATGTTAAGAAATGCATTAATCATTGAAGAT240    ACTGGAGATAACAACGTTGTTAAAATTGGTAAAACAGTAACGTTTGTAGAATTACCAGGT300    GATGAAGAGGAAAGTTATCAAATCGTTGGTTCAGCTGAATCAGATGCATTTAATGGTAAG360    ATTTCAAATGAATCACCAATGGCTAAAGCGTTAATTGGTAAAGGTTTAGATGATGAAGTT420    CGTGTTCCACTACCTAATGGTGGCGAAATGAACGTNAAAATTGTTAATATCCAATAA477    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 158 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetGluAsnHisLysGlnTyrProMetThrGlnGluSerPheGluLys    151015    LeuGluArgGluLeuGluGluLeuLysThrValLysArgProGluVal    202530    ValGluLysIleLysValAlaArgSerPheGlyAspLeuSerGluAsn    354045    SerGluTyrAspAlaAlaLysAspGluGlnGlyPheIleGluGlnAsp    505560    IleGlnArgIleGluHisMetLeuArgAsnAlaLeuIleIleGluAsp    65707580    ThrGlyAspAsnAsnValValLysIleGlyLysThrValThrPheVal    859095    GluLeuProGlyAspGluGluGluSerTyrGlnIleValGlySerAla    100105110    GluSerAspAlaPheAsnGlyLysIleSerAsnGluSerProMetAla    115120125    LysAlaLeuIleGlyLysGlyLeuAspAspGluValArgValProLeu    130135140    ProAsnGlyGlyGluMetAsnValLysIleValAsnIleGln    145150155    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 363 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ATGGAAAATCACAAGCAATATCCAATGACTCAAGAAAGTTTTGAAAAATTAGAGCGTGAA60    CTTGAACAATTAAAAACAGTTAAGCGTCCTGAAGTTGTAGAGAACATTAAAGTTGCCCGT120    TCATTTGGTGACTTATCAGAGAACTCTGAGTATGATGCCGCAAAAGATGAACAAGGATTC180    TTCGAACAAGATATTCTGAGAATTGAGCATATGTCAAGAAATGCACTAATCATGAAGATA240    CTGGAGATAACAACGTTGTTAAAAATTGGGAAAACAGTAACGTTTGTTCAATTCACAGGT300    GATGAACACGAACATTACCCAATCGTTGGGTCAACTGAATCCAAATCCTTTAATGGTAAG360    ATT363    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetGluAsnHisLysGlnTyrProMetThrGlnGluSerPheGluLys    151015    LeuGluArgGluLeuGluGlnLeuLysThrValLysArgProGluVal    202530    ValGluAsnIleLysValAlaArgSerPheGlyAspLeuSerGluAsn    354045    SerGluTyrAspAlaAlaLysAspGluGlnGlyPhePheGluGlnAsp    505560    IleLeuArgIleGluHisMetSerArgAsnAlaLeuIleMetLysIle    65707580    LeuGluIleThrThrLeuLeuLysIleGlyLysThrValThrPheVal    859095    GlnPheThrGlyAspGluHisGluHisTyrProIleValGlySerGlu    100105110    SerLysSerPheAsnGlyLysIle    115120    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GGAATTCATGGAAAATCACAAGCAATATCCAAT33    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GGAATTCATCATACTCAGAGTTCTCTGATAAGTC34    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding SEQID NO:2.
 2. An isolated polynucleotide segment comprising a nucleotidesequence which is fully complementary to the polynucleotide segment ofclaim
 1. 3. An isolated vector comprising the polynucleotide segment ofclaim
 1. 4. An isolated vector comprising the polynucleotide segment ofclaim
 2. 5. An isolated host cell comprising the vector of claim
 3. 6.An isolated host cell comprising the vector of claim
 4. 7. A process forproducing a greA polypeptide comprising the step of culturing the hostcell of claim 5 under conditions sufficient for the production of saidpolypeptide.
 8. An isolated polynucleotide segment comprising SEQ IDNO:1.
 9. An isolated polynucleotide segment comprising a nucleotidesequence from position 1 to 474 inclusive of the polynucleotide sequenceset forth in SEQ ID NO:1.
 10. An isolated polynucleotide segmentcomprising a nucleotide sequence which is fully complementary to thepolynucleotide segment of claim 8 or
 9. 11. An isolated vectorcomprising the polynucleotide segment of claim 8 or
 9. 12. An isolatedvector comprising the polynucleotide segment of claim
 10. 13. Anisolated host cell comprising the vector of claim
 11. 14. An isolatedhost cell comprising the vector of claim
 12. 15. A process for producinga greA polypeptide comprising the step of culturing the host cell ofclaim 13 under conditions sufficient for the production of saidpolypeptide.